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Disulfide-rich peptides such as defensins play diverse roles in immunity and ion channel modulation, as well as constituting the bioactive components of many animal venoms. We investigated the structure and bioactivity of U-RDTX-Pp19, a peptide previously discovered in venom of the assassin bug Pristhesancus plagipennis. Recombinant Pp19 (rPp19) was found to possess insecticidal activity when injected into Drosophila melanogaster. A bioinformatic search revealed that domains homologous to Pp19 are produced by assassin bugs and diverse other arthropods. rPp19 co-eluted with native Pp19 isolated from P. plagipennis, which we found is more abundant in hemolymph than venom. We solved the three-dimensional structure of rPp19 using 2D 1 H NMR spectroscopy, finding that it adopts a disulfide-stabilized structure highly similar to known trans-defensins, with the same cystine connectivity as human α-defensin (I-VI, II-IV, and III-V). The structure of Pp19 is unique among reported structures of arthropod peptides., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Gene duplication followed by nucleotide differentiation is one of the simplest mechanisms to develop new functions for genes. However, the evolutionary processes underlying the divergence of multigene families remain controversial. We used multigene families found within the diversity of toxic proteins in centipede venom to test two hypotheses related to venom evolution: the two-speed mode of venom evolution and the rapid accumulation of variation in exposed residues (RAVER) model. The two-speed mode of venom evolution proposes that different types of selection impact ancient and younger venomous lineages with negative selection being the predominant form in ancient lineages and positive selection being the dominant form in younger lineages. The RAVER hypothesis proposes that, instead of different types of selection acting on different ages of venomous lineages, the different types of selection will selectively contribute to amino acid variation based on whether the residue is exposed to the solvent where it can potentially interact directly with toxin targets. This hypothesis parallels the longstanding understanding of protein evolution that suggests that residues found within the structural or active regions of the protein will be under negative or purifying selection, and residues that do not form part of these areas will be more prone to positive selection. To test these two hypotheses, we compared the venom of 26 centipedes from the order Scolopendromorpha from six currently recognized species from across North America using both transcriptomics and proteomics. We first estimated their phylogenetic relationships and uncovered paraphyly among the genus Scolopendra and evidence for cryptic diversity among currently recognized species. Using our phylogeny, we then characterized the diverse venom components from across the identified clades using a combination of transcriptomics and proteomics. We conducted selection-based analyses in the context of predicted three-dimensional properties of the venom proteins and found support for both hypotheses. Consistent with the two-speed hypothesis, we found a prevalence of negative selection across all proteins. Consistent with the RAVER hypothesis, we found evidence of positive selection on solvent-exposed residues, with structural and less-exposed residues showing stronger signal for negative selection. Through the use of phylogenetics, transcriptomics, proteomics, and selection-based analyses, we were able to describe the evolution of venom from an ancient venomous lineage and support principles of protein evolution that directly relate to multigene family evolution., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The identification of novel toxins from overlooked and taxonomically exceptional species bears potential for various pharmacological applications. The remipede Xibalbanus tulumensis, an underwater cave-dwelling crustacean, is the only crustacean for which a venom system has been described. Its venom contains several xibalbin peptides that have an inhibitor cysteine knot (ICK) scaffold., Results: Our screenings revealed that all tested xibalbin variants particularly inhibit potassium channels. Xib 1 and xib 13 with their eight-cysteine domain similar to spider knottins also inhibit voltage-gated sodium channels. No activity was noted on calcium channels. Expanding the functional testing, we demonstrate that xib 1 and xib 13 increase PKA-II and Erk1/2 sensitization signaling in nociceptive neurons, which may initiate pain sensitization. Our phylogenetic analysis suggests that xib 13 either originates from the common ancestor of pancrustaceans or earlier while xib 1 is more restricted to remipedes. The ten-cysteine scaffolded xib 2 emerged from xib 1 , a result that is supported by our phylogenetic and machine learning-based analyses., Conclusions: Our functional characterization of synthesized variants of xib 1 , xib 2 , and xib 13 elucidates their potential as inhibitors of potassium channels in mammalian systems. The specific interaction of xib 2 with Kv1.6 channels, which are relevant to treating variants of epilepsy, shows potential for further studies. At higher concentrations, xib 1 and xib 13 activate the kinases PKA-II and ERK1/2 in mammalian sensory neurons, suggesting pain sensitization and potential applications related to pain research and therapy. While tested insect channels suggest that all probably act as neurotoxins, the biological function of xib 1 , xib 2, and xib 13 requires further elucidation. A novel finding on their evolutionary origin is the apparent emergence of X. tulumensis-specific xib 2 from xib 1 . Our study is an important cornerstone for future studies to untangle the origin and function of these enigmatic proteins as important components of remipede but also other pancrustacean and arthropod venoms., (© 2024. The Author(s).)

Background: Evolution of novelty is a central theme in evolutionary biology, yet studying the origins of traits with an apparently discontinuous origin remains a major challenge. Venom systems are a well-suited model for the study of this phenomenon because they capture several aspects of novelty across multiple levels of biological complexity. However, while there is some knowledge on the evolution of individual toxins, not much is known about the evolution of venom systems as a whole. One way of shedding light on the evolution of new traits is to investigate less specialised serial homologues, i.e. repeated traits in an organism that share a developmental origin. This approach can be particularly informative in animals with repetitive body segments, such as centipedes., Results: Here, we investigate morphological and biochemical aspects of the defensive telopodal glandular organs borne on the posterior legs of venomous stone centipedes (Lithobiomorpha), using a multimethod approach, including behavioural observations, comparative morphology, proteomics, comparative transcriptomics and molecular phylogenetics. We show that the anterior venom system and posterior telopodal defence system are functionally convergent serial homologues, where one (telopodal defence) represents a model for the putative early evolutionary state of the other (venom). Venom glands and telopodal glandular organs appear to have evolved from the same type of epidermal gland (four-cell recto-canal type) and while the telopodal defensive secretion shares a great degree of compositional overlap with centipede venoms in general, these similarities arose predominantly through convergent recruitment of distantly related toxin-like components. Both systems are composed of elements predisposed to functional innovation across levels of biological complexity that range from proteins to glands, demonstrating clear parallels between molecular and morphological traits in the properties that facilitate the evolution of novelty., Conclusions: The evolution of the lithobiomorph telopodal defence system provides indirect empirical support for the plausibility of the hypothesised evolutionary origin of the centipede venom system, which occurred through functional innovation and gradual specialisation of existing epidermal glands. Our results thus exemplify how continuous transformation and functional innovation can drive the apparent discontinuous emergence of novelties on higher levels of biological complexity., (© 2024. The Author(s).)

Objectives: To isolate a potassium ion channel Kv4.1 inhibitor from centipede venom, and to determine its sequence and structure., Methods: Ion-exchange chromatography and reversed-phase high-performance liquid chromatography were performed to separate and purify peptide components of centipede venom, and their inhibiting effect on Kv4.1 channel was determined by whole-cell patch clamp recording. The molecular weight of isolated peptide Kv4.1 channel inhibitor was identified with matrix assisted laser desorption ionization-time-of-flight mass spectrometry; its primary sequence was determined by Edman degradation sequencing and two-dimensional mass spectrometry; its structure was established based on iterative thread assembly refinement online analysis., Results: A peptide SsTx-P2 was separated from centipede venom with the molecular weight of 6122.8, and its primary sequence consists of 53 amino acid residues NH 2 -ELTWDFVRTCCKLFPDKSECTKACATEFTGGDESRLKDVWPRKLRSGDSRLKD-OH. Peptide SsTx-P2 potently inhibited the current of Kv4.1 channel transiently transfected in HEK293 cell, with 1.0 μmol/L SsTx-P2 suppressing 95% current of Kv4.1 channel. Its structure showed that SsTx-P2 shared a conserved helical structure., Conclusions: The study has isolated a novel peptide SsTx-P2 from centipede venom, which can potently inhibit the potassium ion channel Kv4.1 and displays structural conservation.

The pathophysiology of recurrent pregnancy loss (RPL) involves deficiencies in the proliferation and migration capacities of endometrial stromal cells (hESCs), which impair embryo implantation and development. Since animal venoms are rich source of bioactive molecules, we aimed to characterize the cytoprotective effects of Lonomia obliqua venom on hESCs. hESCs were isolated from endometrial biopsies and the mechanisms of L. obliqua venomous secretions on cell viability, proliferation and migration were characterized. Venom components were identified by chromatography and proteomic analyses. L. obliqua venom induced hESC proliferation, viability and migration in a dose-dependent manner, both in the presence and absence of serum. By ion-exchange chromatography, one fraction enriched in cytoprotective components and devoid of hemotoxins was obtained. Venom proteome identified at least six protein classes with potential cytoprotective properties (hemolins, lipocalins, hemocyannins, antiviral proteins, antimicrobial peptides, and protease inhibitors). L. obliqua venom protected hESCs from oxidative insult. Cytoprotection was also related to nitric oxide and PKC-ERK-activation and down-regulation of cAMP-PKA-dependent pathways that control cell proliferation. L. obliqua venom-induced hESC viability, proliferation and migration occurs mainly by protecting against oxidative damage and activating ERK. Thus, L. obliqua venom components are promising pharmacological tools to understand the underlying mechanisms of hESC deficiency in RPL., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Research on centipede venoms has led to the discovery of a diverse array of novel proteins and peptides, including those with homology to previously discovered toxin families (e.g., phospholipase A2s and pM12a metalloproteases) and novel toxin families not previously detected in venoms (e.g., β-pore forming toxins and scoloptoxins). Most of this research has focused on centipedes in the order Scolopendromorpha, particularly those in the families Scolopendridae, Cryptopidae, and Scolopocryptopidae. To generate the first high-throughput venom characterization for a centipede in the scolopendromorph family Plutoniumidae, we performed venom-gland transcriptomics and venom proteomics on two Theatops posticus. We identified a total of 64 venom toxins, 60 of which were detected in both the venom-gland transcriptome and venom proteome and four of which were only detected transcriptomically. We detected a single highly abundant arylsulfatase B (ARSB) toxin, the first ARSB toxin identified from centipede venoms. As ARSBs have been detected in other venomous species (e.g., scorpions), ARSBs in T. posticus highlights a new case of convergent evolution across venoms. Theatops posticus venom also contained a much higher abundance and diversity of phospholipase A2 toxins compared to other characterized centipede venoms. Conversely, we detected other common centipedes toxins, such as CAPs and scoloptoxins, at relatively low abundances and diversities. Our observation of a diverse set of toxins from T. posticus venom, including those from novel toxin families, emphasizes the importance of studying unexplored centipede taxonomic groups and the continued potential of centipede venoms for novel toxin discovery and unraveling the molecular mechanisms underlying trait evolution., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Darin R. Rokyta reports financial support was provided by National Science Foundation. Darin R. Rokyta reports a relationship with National Science Foundation that includes: funding grants., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Venomous animals utilize venom glands to secrete and store powerful toxins for intraspecific and/or interspecific antagonistic interactions, implying that tissue-specific resistance is essential for venom glands to anatomically separate toxins from other tissues. Here, we show the mechanism of tissue-specific resistance in centipedes (Scolopendra subspinipes mutilans), where the splice variant of the receptor repels its own toxin. Unlike the well-known resistance mechanism by mutation in a given exon, we found that the KCNQ1 channel is highly expressed in the venom gland as a unique splice variant in which the pore domain and transmembrane domain six, partially encoded by exon 6 (rather than 7 as found in other tissues), contain eleven mutated residues. Such a splice variant is sufficient to gain resistance to SsTx (a lethal toxin for giant prey capture) in the venom gland due to a partially buried binding site. Therefore, the tissue-specific KCNQ1 modification confers resistance to the toxins, establishing a safe zone in the venom-storing/secreting environment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Venoms are a complex cocktail of biologically active molecules, including peptides, proteins, polyamide, and enzymes widely produced by venomous organisms. Through long-term evolution, venomous animals have evolved highly specific and diversified peptides and proteins targeting key physiological elements, including the nervous, blood, and muscular systems. Centipedes are typical venomous arthropods that rely on their toxins primarily for predation and defense. Although centipede bites are frequently reported, the composition and effect of centipede venoms are far from known. With the development of molecular biology and structural biology, the research on centipede venoms, especially peptides and proteins, has been deepened. Therefore, we summarize partial progress on the exploration of the bioactive peptides and proteins in centipede venoms and their potential value in pharmacological research and new drug development.

Nineteen U.S. allergen extracts were standardized by the U.S. Food and Drug Administration (FDA) between 1987 and 1998, including of two house-dust mites, short ragweed, cat hair and cat pelt, seven temperate and one southern grass, and six Hymenoptera venom preparations. Relevant literature was reviewed. For each allergen, a "representative" extract was established; the potency of each representative extract was determined by measurement of the total protein content (Hymenoptera venom), radial diffusion measurement of the dominant allergen (short ragweed and cat), or, if there was no dominant allergen, then by quantitative skin testing by using the ID 50 EAL (intradermal dilution for 50 mm sum of erythema determines the bioequivalent allergy units) method. In vitro tests were developed to allow the manufacturer to demonstrate that each lot of its extract was statistically identical, within defined limits, to the FDA reference extract. These tests included radial immunodiffusion, competitive enzyme-linked immunosorbent assay, and isoelectric focusing. The standardized extracts offer the advantage of consistent potency from lot to lot for each manufacturer and also from manufacturer to manufacturer, and assure the presence of recognized significant allergens within the extract. Therefore, standardized extracts offer improved safety and efficacy over their nonstandardized predecessors.

Centipedes are one of the most ancient and successful living venomous animals. They have evolved spooky venoms to deter predators or hunt prey, and are widely distributed throughout the world besides Antarctica. Neurotoxins are the most important virulence factor affecting the function of the nervous system. Ion channels and receptors expressed in the nervous system, including Na V , K V , Ca V , and TRP families, are the major targets of peptide neurotoxins. Insight into the mechanism of neurotoxins acting on ion channels contributes to our understanding of the function of both channels and centipede venoms. Meanwhile, the novel structure and selective activities give them the enormous potential to be modified and exploited as research tools and biological drugs. Here, we review the centipede venom peptides that act on ion channels.

Linear cationic venom peptides are antimicrobial peptides (AMPs) that exert their effects by damaging cell membranes. These peptides can be highly specific, and for some, a significant therapeutic value was proposed, in particular for treatment of bacterial infections. A prolific source of novel AMPs are arthropod venoms, especially those of hitherto neglected groups such as pseudoscorpions. In this study, we describe for the first time pharmacological effects of AMPs discovered in pseudoscorpion venom. We examined the antimicrobial, cytotoxic, and insecticidal activity of full-length Checacin1, a major component of the Chelifer cancroides venom, and three truncated forms of this peptide. The antimicrobial tests revealed a potent inhibitory activity of Checacin1 against several bacteria and fungi, including methicillin resistant Staphylococcus aureus (MRSA) and even Gram-negative pathogens. All peptides reduced survival rates of aphids, with Checacin1 and the C-terminally truncated Checacin1 1-21 exhibiting effects comparable to Spinosad, a commercially used pesticide. Cytotoxic effects on mammalian cells were observed mainly for the full-length Checacin1. All tested peptides might be potential candidates for developing lead structures for aphid pest treatment. However, as these peptides were not yet tested on other insects, aphid specificity has not been proven. The N- and C-terminal fragments of Checacin1 are less potent against aphids but exhibit no cytotoxicity on mammalian cells at the tested concentration of 100 µM.

Among the Chilopoda class of centipede, the Cryptops genus is one of the most associated with envenomation in humans in the metropolitan region of the state of São Paulo. To date, there is no study in the literature about the toxins present in its venom. Thus, in this work, a transcriptomic characterization of the Cryptops iheringi venom gland, as well as a proteomic analysis of its venom, were performed to obtain a toxin profile of this species. These methods indicated that 57.9% of the sequences showed to be putative toxins unknown in public databases; among them, we pointed out a novel putative toxin named Cryptoxin-1. The recombinant form of this new toxin was able to promote edema in mice footpads with massive neutrophils infiltration, linking this toxin to envenomation symptoms observed in accidents with humans. Our findings may elucidate the role of this toxin in the venom, as well as the possibility to explore other proteins found in this work.

Discriminating Polistes dominula and Vespula spp. venom allergy is of growing importance worldwide, as systemic reactions to either species' sting can lead to severe outcomes. Administering the correct allergen-specific immunotherapy is therefore a prerequisite to ensure the safety and health of venom-allergic patients. Component-resolved diagnostics of Hymenoptera venom allergy might be improved by adding additional allergens to the diagnostic allergen panel. Therefore, three potential new allergens from P. dominula venom-immune responsive protein 30 (IRP30), vascular endothelial growth factor C (VEGF C) and phospholipase A2 (PLA2)-were cloned, recombinantly produced and biochemically characterized. Sera sIgE titers of Hymenoptera venom-allergic patients were measured in vitro to assess the allergenicity and potential cross-reactivity of the venom proteins. IRP30 and VEGF C were classified as minor allergens, as sensitization rates lay around 20-40%. About 50% of P. dominula venom-allergic patients had measurable sIgE titers directed against PLA2 from P. dominula venom. Interestingly, PLA2 was unable to activate basophils of allergic patients, questioning its role in the context of clinically relevant sensitization. Although the obtained results hint to a questionable benefit of the characterized P. dominula venom proteins for improved diagnosis of venom-allergic patients, they can contribute to a deeper understanding of the molecular mechanisms of Hymenoptera venoms and to the identification of factors that determine the allergenic potential of proteins.

Velvet ants (Hymenoptera: Mutillidae) are a family of solitary parasitoid wasps that are renowned for their painful stings. We explored the chemistry underlying the stings of mutillid wasps of the genus Dasymutilla Ashmead. Detailed analyses of the venom composition of five species revealed that they are composed primarily of peptides. We found that two kinds of mutillid venom peptide appear to be primarily responsible for the painful effects of envenomation. These same peptides also have defensive utility against invertebrates, since they were able to incapacitate and kill honeybees. Both act directly on cell membranes where they directly increase ion conductivity. The defensive venom peptides of Dasymutilla bear a striking similarity, in structure and mode of action, to those of the ant Myrmecia gulosa (Fabricius), suggesting either retention of ancestral toxins, or convergence driven by similar life histories and defensive selection pressures. Finally, we propose that other highly expressed Dasymutilla venom peptides may play a role in parasitisation, possible in delay or arrest of host development. This study represents the first detailed account of the composition and function of the venoms of the Mutillidae.

Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Doratifera vulnerans Contrary to dogma that defensive venoms are simple in composition, D. vulnerans produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides <10 kDa. Three of the most abundant families of venom peptides (vulnericins) are 1) analogs of the adipokinetic hormone/corazonin-related neuropeptide, some of which are picomolar agonists of the endogenous insect receptor; 2) linear cationic peptides derived from cecropin, an insect innate immune peptide that kills bacteria and parasites by disrupting cell membranes; and 3) disulfide-rich knottins similar to those that dominate spider venoms. Using venom fractionation and a suite of synthetic venom peptides, we demonstrate that the cecropin-like peptides are responsible for the dominant pain effect observed in mammalian in vitro and in vivo nociception assays and therefore are likely to cause pain after natural envenomations by D. vulnerans Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides., Competing Interests: The authors declare no competing interest.

Assassin bugs are venomous insects that prey on other arthropods. Their venom has lethal, paralytic, and liquifying effects when injected into prey, but the toxins responsible for these effects are unknown. To identify bioactive assassin bug toxins, venom was harvested from the red tiger assassin bug ( Havinthus rufovarius ), an Australian species whose venom has not previously been characterised. The venom was fractionated using reversed-phase high-performance liquid chromatography, and four fractions were found to cause paralysis and death when injected into sheep blowflies ( Lucilia cuprina ). The amino acid sequences of the major proteins in two of these fractions were elucidated by comparing liquid chromatography/tandem mass spectrometry data with a translated venom-gland transcriptome. The most abundant components were identified as a solitary 12.8 kDa CUB (complement C1r/C1s, Uegf, Bmp1) domain protein and a 9.5 kDa cystatin. CUB domains are present in multidomain proteins with diverse functions, including insect proteases. Although solitary CUB domain proteins have been reported to exist in other heteropteran venoms, such as that of the bee killer assassin bug Pristhesancus plagipennis , their function is unknown, and they have not previously been reported as lethal or paralysis-inducing. Cystatins occur in the venoms of spiders and snakes, but again with an unknown function. Reduction and alkylation experiments revealed that the H. rufovarius venom cystatin featured five cysteine residues, one of which featured a free sulfhydryl group. These data suggest that solitary CUB domain proteins and/or cystatins may contribute to the insecticidal activity of assassin bug venom.

Allergic reactions to stings of Hymenoptera species can have serious or even fatal consequences. If the identification of the culprit insect is possible, venom-specific immunotherapy effectively cures Hymenoptera venom allergies. Although component-resolved diagnostics has strongly evolved in recent years, the differentiation between allergies to closely related species such as Polistes dominula and Vespula spp. is still challenging. In order to generate the basis for new diagnostic and therapeutic strategies, this study aims at resolving the venom proteomes (venomes) of these species. The venoms of P. dominula and Vespula spp. ( V. germanica , V. vulgaris ) were analyzed by liquid chromatography-mass spectrometry. Resulting proteins were characterized regarding their function, localization and biochemical properties. The analyses yielded 157 proteins in Vespula spp. and 100 in P. dominula venom; 48 proteins, including annotated allergens, were found in both samples. In addition to a variety of venom trace molecules, new allergen candidates such as icarapin-like protein and phospholipase A2 were identified. This study elucidates the venomes of closely related allergy-eliciting Hymenoptera species. The data indicates that relying on marker allergens to differentiate between P. dominula and Vespula spp. venom allergy is probably insufficient and that strategies using cross-reactive major allergens could be more promising.

Insect venom peptides (IVPs) eumenitin, lasiocepsin, lycosin1, mastoparanB, panurgine1, and protonectin possess antibacterial properties, and the ubiquitous enzyme ATP synthase has a peptide-binding site. In the present study, we studied the effect of IVPs on binding and inhibition of three Escherichia coli strains (wild type, mutant, and null) and isolated E. coli ATP synthase. IVPs and their C-terminal amide (-NH 2 ) analogs caused variable inhibition of membrane-bound F 1 F o ATP synthase. While wild type E. coli growth was substantially hampered, null E. coli growth was near normal in the presence of IVPs and their C-terminal-NH 2 analogs. The presence of C-terminal-NH 2 groups on IVPs resulted in increased inhibition of ATP synthase and reduced growth of E. coli strains. Insignificant inhibition of the βDELSEED-motif mutant enzyme with the βAAAAAAA-motif confirmed that IVPs interact with the βDELSEED-motif, also known as the peptide-binding site. The higher level of growth loss in E. coli strains by eumenitin, lasiocepsin, lycosin1, mastoparanB, panurgine1, and protonectin and their C-terminal-NH 2 analogs suggested the likelihood of additional cellular or molecular targets. IVPs caused inhibition of E. coli strains, which demonstrates an association between antimicrobial traits of IVPs and bacterial ATP synthase., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Centipedes are among the most ancient groups of venomous predatory arthropods. Extant species belong to five orders, but our understanding of the composition and evolution of centipede venoms is based almost exclusively on one order, Scolopendromorpha. To gain a broader and less biased understanding we performed a comparative proteotranscriptomic analysis of centipede venoms from all five orders, including the first venom profiles for the orders Lithobiomorpha, Craterostigmomorpha, and Geophilomorpha. Our results reveal an astonishing structural diversity of venom components, with 93 phylogenetically distinct protein and peptide families. Proteomically-annotated gene trees of these putative toxin families show that centipede venom composition is highly dynamic across macroevolutionary timescales, with numerous gene duplications as well as functional recruitments and losses of toxin gene families. Strikingly, not a single family is found in the venoms of representatives of all five orders, with 67 families being unique for single orders. Ancestral state reconstructions reveal that centipede venom originated as a simple cocktail comprising just four toxin families, with very little compositional evolution happening during the approximately 50 My before the living orders had diverged. Venom complexity then increased in parallel within the orders, with scolopendromorphs evolving particularly complex venoms. Our results show that even venoms composed of toxins evolving under the strong constraint of negative selection can have striking evolutionary plasticity on the compositional level. We show that the functional recruitments and losses of toxin families that shape centipede venom arsenals are not concentrated early in their evolutionary history, but happen frequently throughout., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Assassin bugs (Reduviidae) produce venoms that are insecticidal, and which induce pain in predators, but the composition and function of their individual venom components is poorly understood. We report findings on the venom system of the red-spotted assassin bug Platymeris rhadamanthus , a large species of African origin that is unique in propelling venom as a projectile weapon when threatened. We performed RNA sequencing experiments on venom glands (separate transcriptomes of the posterior main gland, PMG, and the anterior main gland, AMG), and proteomic experiments on venom that was either defensively propelled or collected from the proboscis in response to electrostimulation. We resolved a venom proteome comprising 166 polypeptides. Both defensively propelled venom and most venom samples collected in response to electrostimulation show a protein profile similar to the predicted secretory products of the PMG, with a smaller contribution from the AMG. Pooled venom samples induce calcium influx via membrane lysis when applied to mammalian neuronal cells, consistent with their ability to cause pain when propelled into the eyes or mucus membranes of potential predators. The same venom induces rapid paralysis and death when injected into fruit flies. These data suggest that the cytolytic, insecticidal venom used by reduviids to capture prey is also a highly effective defensive weapon when propelled at predators., Competing Interests: The authors declare no conflict of interest.

Sexually dimorphic traits are widespread across metazoans and are often the result of sex-specific inheritance or sex-based differences in gene expression. Intersexual differences have even been observed in invertebrate venoms, although the identification of these differences has been limited to the more well-studied groups, such as scorpions and spiders, where sex-based differences in morphology and behavior are apparent. Recent studies on centipede venom have identified evidence of intraspecific variation, but intersexual differences have not been reported. To investigate the potential for sex-based differences in centipede venom composition, we performed reversed-phase high performance liquid chromatography (RP-HPLC) analyses on five male and 15 female eastern bark centipedes (Hemiscolopendra marginata) from the Apalachicola National Forest in northern Florida. After detecting a significant sex-based difference in H. marginata venom composition, we completed a high-throughput venom-gland transcriptomic and venom proteomic analysis of one male and one female to determine the genetic basis for differences in venom composition. We identified 47 proteomically confirmed toxins and 717 nontoxin transcripts in H. marginata venom-glands. Of these proteomically confirmed toxins, the most abundantly expressed in the male venom included ion channel-modulating toxins and toxins so divergent from any characterized homologs that they could not be given a functional classification, whereas the most abundantly expressed in the female venom were γ-glutamyl transferases and CAPs (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins). These differences were then confirmed by performing replicate LC-MS/MS analyses on the venom from an additional three male and three female H. marginata. Our RP-HPLC and high-throughput transcriptomic and proteomic approach resulted in not only an in-depth characterization of H. marginata venom, but represents the first example of sex-based variation in centipede venoms., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Disulfide-rich peptides (DRPs) play diverse physiological roles and have emerged as attractive sources of pharmacological tools and drug leads. Here we describe the 3D structure of a centipede venom peptide, U-SLPTX 15 -Sm2a, whose family defines a unique class of one of the most widespread DRP folds known, the cystine-stabilized α/β fold (CSαβ). This class, which we have named the two-disulfide CSαβ fold (2ds-CSαβ), contains only two internal disulfide bonds as opposed to at least three in all other confirmed CSαβ peptides, and constitutes one of the major neurotoxic peptide families in centipede venoms. We show the 2ds-CSαβ is widely distributed outside centipedes and is likely an ancient fold predating the split between prokaryotes and eukaryotes. Our results provide insights into the ancient evolutionary history of a widespread DRP fold and highlight the usefulness of 3D structures as evolutionary tools., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The millions of extant arthropod species are testament to their evolutionary success that can at least partially be attributed to venom usage, which evolved independently in at least 19 arthropod lineages. While some arthropods primarily use venom for predation (e.g., spiders and centipedes) or defense (e.g., bees and caterpillars), it can also have more specialised functions (e.g. in parasitoid wasps to paralyse arthropods for their brood to feed on) or even a combination of functions (e.g. the scorpion Parabuthus transvaalicus can deliver a prevenom for predator deterrence and a venom for predation). Most arthropod venoms are complex cocktails of water, salts, small bioactive molecules, peptides, enzymes and larger proteins, with peptides usually comprising the majority of toxins. Some spider venoms have been reported to contain >1000 peptide toxins, which function as combinatorial libraries to provide an evolutionary advantage. The astounding diversity of venomous arthropods multiplied by their enormous toxin arsenals results in an almost infinite resource for novel bioactive molecules. The main challenge for exploiting this resource is the small size of most arthropods, which can be a limitation for current venom extraction techniques. Fortunately, recent decades have seen an incredible improvement in transcriptomic and proteomic techniques that have provided increasing sensitivity while reducing sample requirements. In turn, this has provided a much larger variety of arthropod venom compounds for potential applications such as therapeutics, molecular probes for basic research, bioinsecticides or anti-parasitic drugs. This special issue of Toxicon aims to cover the breadth of arthropod venom research, including toxin evolution, pharmacology, toxin discovery and characterisation, toxin structures, clinical aspects, and potential applications., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Comprising of over a million described species of highly diverse invertebrates, Arthropoda is amongst the most successful animal lineages to have colonized aerial, terrestrial, and aquatic domains. Venom, one of the many fascinating traits to have evolved in various members of this phylum, has underpinned their adaptation to diverse habitats. Over millions of years of evolution, arthropods have evolved ingenious ways of delivering venom in their targets for self-defence and predation. The morphological diversity of venom delivery apparatus in arthropods is astounding, and includes extensively modified pedipalps, tail (telson), mouth parts (hypostome), fangs, appendages (maxillulae), proboscis, ovipositor (stinger), and hair (urticating bristles). Recent investigations have also unravelled an astonishing venom biocomplexity with molecular scaffolds being recruited from a multitude of protein families. Venoms are a remarkable bioresource for discovering lead compounds in targeted therapeutics. Several components with prospective applications in the development of advanced lifesaving drugs and environment friendly bio-insecticides have been discovered from arthropod venoms. Despite these fascinating features, the composition, bioactivity, and molecular evolution of venom in several arthropod lineages remains largely understudied. This review highlights the prevalence of venom, its mode of toxic action, and the evolutionary dynamics of venom in Arthropoda, the most speciose phylum in the animal kingdom., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Insect toxins comprise a diverse array of chemicals ranging from small molecules, polyamines and peptide toxins. Many target nervous system and neuromuscular ion channels and so rapidly affect the behaviour of animals to which the toxin is applied or injected. Other modes of action have also been identified. Wasps, bees, flies, beetles and ants generate a rich arsenal of channel-active toxins, some of which offer selective pharmacological probes that target particular ion channels, while others act on more than one type of channel. Philanthotoxins from the digger wasp have been fruitful in adding to our understanding of ligand-gated ion channels both in the nervous system and at neuromuscular junctions. Fire ants produce the toxic alkaloid solenopsin, a molecule which has stimulated attempts to generate synthetic compounds with insecticidal activity. Apamin from bee venom targets calcium-activated potassium channels, which can in turn influence the release of neuropeptides. Melittin, another bee venom component, is a membrane-acting peptide. The saliva of the assassin bug contains toxins that target the voltage-gated calcium channels of their insect prey. Certain beetles produce diamphotoxin, a haemolytic peptide toxin with traditional use as an arrow poison and others generate leptinotarsin with similar properties. Mastoparan is a powerful peptide toxin present in the venom of wasps. Its toxic actions can be engineered out leaving a potent antimicrobial molecule of interest. In this short review we describe the actions of selected insect toxins and evaluate their potential as neuroactive pharmacological tools, candidate lead molecules for insect control and therapeutic candidates with potential antimicrobial, antiviral and anti-cancer applications., (Copyright © 2018. Published by Elsevier Inc.)

Assassin flies (Diptera: Asilidae) inject paralysing venom into insect prey during hunting, but their venoms are poorly characterised in comparison to those produced by spiders, scorpions, or hymenopteran insects. Here we investigated the composition of the venom of the giant Australian assassin fly Dolopus genitalis using a combination of insect microinjection assays, calcium imaging assays of mammalian sensory neurons, proteomics and transcriptomics. Injection of venom into blowflies ( Lucilia cuprina ) produced rapid contractile paralysis (PD 50 at 1 min = 3.1 μg per fly) followed by death, and also caused immediate activation of mouse dorsal root ganglion neurons (at 50 ng/μL). These results are consistent with venom use for both prey capture and predator deterrence. Paragon searches of tandem mass spectra of venom against a translated thoracic gland RNA-Seq database identified 122 polypeptides present in the venom, including six linear and 21 disulfide-rich peptides. Some of these disulfide-rich peptides display sequence homology to peptide families independently recruited into other animal venoms, including inhibitor cystine knots, cystine-stabilised α/β defensins, Kazal peptides, and von Willebrand factors. Numerous enzymes are present in the venom, including 35 proteases of the S1 family, proteases of the S10, C1A, M12A, M14, and M17 families, and phosphatase, amylase, hydrolase, nuclease, and dehydrogenase-like proteins. These results highlight convergent molecular evolution between the assassin flies and other venomous animals, as well as the unique and rich molecular composition of assassin fly venom.

Centipede bites are usually characterized by mildly to moderately painful encounters with humans, however, they are relatively infrequent. The vast majority of centipede envenomations do not cause severe symptoms and only in very rare cases more serious symptoms such as myocardial ischemia and infarction, hematuria, hemoglobinuria, rhabdomyolysis, hemorrhage, pruritus, eosinophilic cellulitis, as well as anaphylaxis are observed. More prevalent are symptoms including pain, paresthesia, lethargy, localized necrosis, headache, dizziness and nausea. The numerous symptoms and complications elicited by these envenomations indicate that centipede venom possesses an arsenal of chemical components with functional diversity. Centipede venom is a rich and complex natural source of bioactive proteins, peptides and other small molecules that aid in predation or defense. The venom can induce myotoxic, cardiotoxic, neurotoxic and other toxic effects. The constituents target different cellular processes and pathways which in turn trigger a cascade of physiological reactions in the victim. The venom components are potent and selective on peripheral targets; thus, they are valuable in studying the molecular basis of these envenomation symptoms and complications. This review highlights the clinical importance of centipede envenomation and the recent discoveries on the underlying molecular mechanisms of the resulting symptoms which is crucial in therapy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Envenomation by the larval or pupal stages of moths occurs when the victim presses their hairs. They penetrate the subcutaneous tissue, releasing toxins such as proteolytic enzymes, histamine and other pro-inflammatory substances. Cutaneous reactions, including severe pain, oedema and erythema are frequent local manifestations of caterpillar envenomation, but, in some cases, the reactions can evolve into vesicles, bullae, erosions, petechiae, superficial skin necrosis and ulcerations. Alternatively, some individual can develop allergic reactions, renal failure, osteochondritis, deformity and immobilization of the affected joints and intracerebral bleeding. Caterpillars produce venom to protect themselves from predators; contact with humans is accidental and deserves close attention. Their venoms have not been well studied, except for toxins from some few species. The present review brings together data on venomous caterpillars of moths, primarily addressing the available literature on diversity among the different families that cause accident in humans, the structures used in their defense, venom composition and clinical aspects of the envenomations. Understanding the molecular mechanisms of action of caterpillars' toxins may lead to the development of more adequate treatments., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The limited number of centipede venom characterizations have revealed a rich diversity of toxins, and recent work has suggested centipede toxins may be more rapidly diversifying than previously considered. Additionally, many identified challenges in venomics research, including assembly and annotation methods, toxin quantification, and the ability to provide biological or technical replicates, have yet to be addressed in centipede venom characterizations. We performed high-throughput, quantifiable transcriptomic and proteomic methods on two individual Scolopendra viridis centipedes from North Florida. We identified 39 toxins that were proteomically confirmed, and 481 nontoxins that were expressed in the venom gland of S. viridis. The most abundant toxins expressed in the venom of S. viridis belonged to calcium and potassium ion-channel toxins, venom allergens, metalloproteases, and β-pore forming toxins. We compared our results to the previously characterized S. viridis from Morelos, Mexico, and found only five proteomically confirmed toxins in common to both localities, suggesting either extreme toxin divergence within S. viridis, or that these populations may represent entirely different species. By using multiple assembly and annotation methods, we generated a comprehensive and quantitative reference transcriptome and proteome of a Scolopendromorpha centipede species, while overcoming some of the challenges present in venomics research., (Copyright © 2018. Published by Elsevier Ltd.)

Arthropods are a diverse and ancient group of invertebrate animals, which constitute approximately 75-85% of all known species on earth. Many arthropod species, such as spiders, scorpions and even some crustaceans, contain venoms that can be very complex, representing natural combinatorial libraries of bioactive compounds. Characterization of the compounds in these libraries has helped the development of tools for pharmacology, and the discovery of lead molecules for therapeutic treatments. A critical aspect in the characterization of venom compounds is determination of three-dimensional structure. Structural analysis can provide insight into many processes including understanding target interactions and developing more potent and selective drug leads. Arthropod venoms are an extremely rich source of novel structures and this review provides an overview of this structural diversity, including structures of proteins, peptides and small molecules., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The adenosine triphosphate (ATP)-sensitive (K ATP ) channels in pancreatic β cells couple the blood glucose level to insulin secretion. K ATP channels in pancreatic β cells comprise the pore-forming Kir6.2 and the modulatory sulfonylurea receptor 1 (SUR1) subunits. Currently, there is no high-affinity and relatively specific inhibitor for the Kir6.2 pore. The importance of developing such inhibitors is twofold. First, in many cases, the lack of such an inhibitor precludes an unambiguous determination of the Kir6.2's role in certain physiological and pathological processes. This problem is exacerbated because Kir6.2 knockout mice do not yield the expected phenotypes of hyperinsulinemia and hypoglycemia, which in part, may reflect developmental adaptation. Second, mutations in Kir6.2 or SUR1 that increase the K ATP current cause permanent neonatal diabetes mellitus (PNDM). Many patients who have PNDM have been successfully treated with sulphonylureas, a common class of antidiabetic drugs that bind to SUR1 and indirectly inhibit Kir6.2, thereby promoting insulin secretion. However, some PNDM-causing mutations render K ATP channels insensitive to sulphonylureas. Conceptually, because these mutations are located intracellularly, an inhibitor blocking the Kir6.2 pore from the extracellular side might provide another approach to this problem. Here, by screening the venoms from >200 animals against human Kir6.2 coexpressed with SUR1, we discovered a small protein of 54 residues (SpTx-1) that inhibits the K ATP channel from the extracellular side. It inhibits the channel with a dissociation constant value of 15 nM in a relatively specific manner and with an apparent one-to-one stoichiometry. SpTx-1 evidently inhibits the channel by primarily targeting Kir6.2 rather than SUR1; it inhibits not only wild-type Kir6.2 coexpressed with SUR1 but also a Kir6.2 mutant expressed without SUR1. Importantly, SpTx-1 suppresses both sulfonylurea-sensitive and -insensitive, PNDM-causing Kir6.2 mutants. Thus, it will be a valuable tool to investigate the channel's physiological and biophysical properties and to test a new strategy for treating sulfonylurea-resistant PNDM.

Among venomous animals, Hymenoptera have been suggested as a rich source of natural toxins. Due to their broad ecological diversity, venom from Hymenoptera insects (bees, wasps and ants) have evolved differentially thus widening the types and biological functions of their components. To date, insect toxinology analysis have scarcely uncovered the complex composition of bee, wasp and ant venoms which include low molecular weight compounds, highly abundant peptides and proteins, including several allergens. In Hymenoptera, these complex mixtures of toxins represent a potent arsenal of biological weapons that are used for self-defense, to repel intruders and to capture prey. Consequently, Hymenoptera venom components have a broad range of pharmacological targets and have been extensively studied, as promising sources of new drugs and biopesticides. In addition, the identification and molecular characterization of Hymenoptera venom allergens have allowed for the rational design of component-resolved diagnosis of allergy, finally improving the outcome of venom immunotherapy (VIT). Until recently, a limited number of Hymenoptera venoms had been unveiled due to the technical limitations of the approaches used to date. Nevertheless, the application of novel techniques with high dynamic range has significantly increased the number of identified peptidic and proteinaceous toxins. Considering this, the present review summarizes the current knowledge about the most representative Hymenoptera venom peptides and proteins which are under study for a better understanding of the insect-caused envenoming process and the development of new drugs and biopesticides., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The venom gland is a ubiquitous organ in Hymenoptera. In insect parasitoids, the venom gland has been shown to have multiple functions including regulation of host immune response, host paralysis, host castration and developmental alteration. However, the role played by the venom gland has been mainly studied in parasitoids developing in larval or pupal hosts while little is known for parasitoids developing in insect eggs. We conducted the first extensive characterization of the venom of the endoparasitoid Ooencyrtus telenomicida (Vassiliev), a species that develops in eggs of the stink bug Nezara viridula (L.). In particular we investigated the structure of the venom apparatus, its functional role and conducted a transcriptomic analysis of the venom gland. We found that injection of O. telenomicida venom induces: 1) a melanized-like process in N. viridula host eggs (host-parasitoid interaction), 2) impairment of the larval development of the competitor Trissolcus basalis (Wollaston) (parasitoid-parasitoid interaction). The O. telenomicida venom gland transcriptome reveals a majority of digestive enzymes (peptidases and glycosylases) and oxidoreductases (laccases) among the most expressed genes. The former enzymes are likely to be involved in degradation of the host resources for the specific benefit of the O. telenomicida offspring. In turn, alteration of host resources caused by these enzymes may negatively affect the larval development of the competitor T. basalis. We hypothesize that the melanization process induced by venom injection could be related to the presence of laccases, which are multicopper oxidases that belong to the phenoloxidases group. This work contributed to a better understanding of the venom in insect parasitoids and allowed to identify candidate genes whose functional role can be investigated in future studies., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Component-resolved diagnosis based on the use of well-defined, properly characterised and purified natural and recombinant allergens constitutes a new approach in the diagnosis of venom allergy. Prospective readers may benefit from an up-to-date review on the allergens. The best characterised venom is that of Apis mellifera, whose main allergens are phospholipase A2 (Api m1), hyaluronidase (Api m2) and melittin (Api m4). Additionally, in recent years, new allergens of Vespula vulgaris have been identified and include phospholipase A1 (Ves v1), hyaluronidase (Ves v2) and antigen 5 (Ves v5). Polistes species are becoming an increasing cause of allergy in Europe, although only few allergens have been identified in this venom. In this review, we evaluate the current knowledge about molecular diagnosis in hymenoptera venom allergy., (Copyright © 2017 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.)

Centipedes are one of the oldest venomous animals and use their venoms as weapons to attack prey or protect themselves. Their venoms contain various components with different biomedical and pharmacological properties. However, little attention has been paid to the profiles and diversity of their toxin-like proteins/peptides. In this study, we used a proteotranscriptomic approach to uncover the diversity of centipede toxin-like proteins in Scolopendra subspinipes mutilans Nine hundred twenty-three and 6,736 peptides, which were separately isolated from venom and torso tissues, respectively, were identified by ESI-MS/MS and deduced from their transcriptomes. Finally, 1369 unique proteins were identified in the proteome, including 100 proteins that exhibited overlapping expression in venom and torso tissues. Of these proteins, at least 40 proteins were identified as venom toxin-like proteins. Meanwhile, transcriptome mining identified ∼10-fold more toxin-like proteins and enabled the characterization of the precursor architecture of mature toxin-like peptides. Importantly, combined with proteomic and transcriptomic analyses, 25 toxin-like proteins/peptides (neurotoxins accounted for 50%) were expressed outside the venom gland and involved in gene recruitment processes. These findings highlight the extensive diversity of centipede toxin-like proteins and provide a new foundation for the medical-pharmaceutical use of centipede toxin-like proteins. Moreover, we are the first group to report the gene recruitment activity of venom toxin-like proteins in centipede, similar to snakes., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Predatory robber flies (Diptera, Asilidae) have been suspected to be venomous due to their ability to overpower well-defended prey. However, details of their venom composition and toxin arsenal remained unknown. Here, we provide a detailed characterization of the venom system of robber flies through the application of comparative transcriptomics, proteomics and functional morphology. Our results reveal asilid venoms to be dominated by peptides and non-enzymatic proteins, and that the majority of components in the crude venom is represented by just ten toxin families, which we have named Asilidin1-10. Contrary to what might be expected for a liquid-feeding predator, the venoms of robber flies appear to be rich in novel peptides, rather than enzymes with a putative pre-digestive role. The novelty of these peptides suggests that the robber fly venom system evolved independently from hematophagous dipterans and other pancrustaceans. Indeed, six Asilidins match no other venom proteins, while three represent known examples of peptide scaffolds convergently recruited to a toxic function. Of these, members of Asilidin1 closely resemble cysteine inhibitor knot peptides (ICK), of which neurotoxic variants occur in cone snails, assassin bugs, scorpions and spiders. Synthesis of one of these putative ICKs, U-Asilidin₁-Mar1a, followed by toxicity assays against an ecologically relevant prey model revealed that one of these likely plays a role as a neurotoxin involved in the immobilization of prey. Our results are fundamental to address these insights further and to understand processes that drive venom evolution in dipterans as well as other arthropods., Competing Interests: The authors declare no conflict of interest.

The Australian paralysis tick (Ixodes holocyclus) secretes neuropathic toxins into saliva that induce host paralysis. Salivary glands and viscera were dissected from fully engorged female I. holocyclus ticks collected from dogs and cats with paralysis symptoms. cDNA from both tissue samples were sequenced using Illumina HiSeq 100 bp pair end read technologies. Unique and non-redundant holocyclotoxin sequences were designated as HT2-HT19, as none were identical to the previously described HT1. Specific binding to rat synaptosomes was determined for synthetic HTs, and their neurotoxic capacity was determined by neonatal mouse assay. They induced a powerful paralysis in neonatal mice, particularly HT4 which produced rapid and strong respiratory distress in all animals tested. This is the first known genomic database developed for the Australian paralysis tick. The database contributed to the identification and subsequent characterization of the holocyclotoxin family that will inform the development of novel anti-paralysis control methods., (Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Voltage-gated sodium channel Na V 1.7 serves as an attractive target for chronic pain treatment. Several venom peptides were found to selectively inhibit Na V 1.7 but with intrinsic problems. Among them, Ssm6a, a recently discovered centipede venom peptide, shows the greatest selectivity against Na V 1.7, but dissociates from the target too fast and loses bioactivity in synthetic forms. As a disulfide-rich venom peptide, it is difficult to optimize Ssm6a by artificial mutagenesis and produce the peptide with common industrial manufacturing methods. Here, we developed a novel protein scaffold fusion strategy to address these concerns. Instead of directly mutating Ssm6a, we genetically fused Ssm6a with a protein scaffold engineered from human muscle fatty acid-binding protein. The resultant fusion protein, SP-TOX, maintained the selectivity and potency of Ssm6a upon Na V 1.7 but dissociated from target at least 10 times more slowly. SP-TOX dramatically reduced inflammatory pain in a rat model through DRG-targeted delivery. Importantly, SP-TOX can be expressed cytosolically in Escherichia coli and purified in a cost-effective way. In summary, our study provided the first example of cytosolically expressed fusion protein with high potency and selectivity on Na V 1.7. Our protein scaffold fusion approach may have its broad application in optimizing disulfide-rich venom peptides for therapeutic usage., (© 2016 John Wiley & Sons A/S.)

Pain is a major symptom of many diseases and results in enormous pressures on human body or society. Currently, clinically used analgesic drugs, including opioids and nonsteroidal anti-inflammatory drugs, have adverse reactions, and thus, the development of new types of analgesic drug candidates is urgently needed. Animal venom peptides have proven to have potential as new types of analgesic medicine. In this research, we describe the isolation and characterization of an analgesic peptide from the crude venom of centipede, Scolopendra subspinipes mutilans. The amino acid sequence of this peptide was identical with SsmTX-I that was previously reported as a specific Kv2.1 ion channel blocker. Our results revealed that SsmTX-I was produced by posttranslational processing of a 73-residue prepropeptide. The intramolecular disulfide bridge motifs of SsmTX-I was Cys1-Cys3 and Cys2-Cys4. Functional assay revealed that SsmTX-I showed potential analgesic activities in formalin-induced paw licking, thermal pain, and acetic acid-induced abdominal writhing mice models. Our research provides the first report of cDNA sequences, disulfide motif, successful synthesis, and analgesic potential of SsmTX-I for the development of pain-killing drugs. It indicates that centipede peptide toxins could be a treasure trove for the search of novel analgesic drug candidates. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.)

Background: Hymenoptera stings can cause severe anaphylaxis in untreated venom-allergic patients. A correct diagnosis regarding the relevant species for immunotherapy is often hampered by clinically irrelevant cross-reactivity. In vespid venom allergy, cross-reactivity between venoms of different species can be a diagnostic challenge. To address immunological IgE cross-reactivity on molecular level, seven recombinant antigens 5 of the most important Vespoidea groups were assessed by different diagnostic setups., Methods: The antigens 5 of yellow jackets, hornets, European and American paper wasps, fire ants, white-faced hornets, and Polybia wasps were recombinantly produced in insect cells, immunologically and structurally characterized, and their sIgE reactivity assessed by ImmunoCAP, ELISA, cross-inhibition, and basophil activation test (BAT) in patients with yellow jacket or Polistes venom allergy of two European geographical areas., Results: All recombinant allergens were correctly folded and structural models and patient reactivity profiles suggested the presence of conserved and unique B-cell epitopes. All antigens 5 showed extensive cross-reactivity in sIgE analyses, inhibition assays, and BAT. This cross-reactivity was more pronounced in ImmunoCAP measurements with venom extracts than in sIgE analyses with recombinant antigens 5. Dose-response curves with the allergens in BAT allowed a differentiated individual dissection of relevant sensitization., Conclusions: Due to extensive cross-reactivity in various diagnostic settings, antigens 5 are inappropriate markers for differential sIgE diagnostics in vespid venom allergy. However, the newly available antigens 5 from further vespid species and the combination of recombinant allergen-based sIgE measurements with BAT represents a practicable way to diagnose clinically relevant sensitization in vespid venom allergy., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: Centipedes are one of the oldest and most successful lineages of venomous terrestrial predators. Despite their use for centuries in traditional medicine, centipede venoms remain poorly studied. However, recent work indicates that centipede venoms are highly complex chemical arsenals that are rich in disulfide-constrained peptides that have novel pharmacology and three-dimensional structure. Areas covered: This review summarizes what is currently known about centipede venom proteins, with a focus on disulfide-rich peptides that have novel or unexpected pharmacology that might be useful from a therapeutic perspective. The authors also highlight the remarkable diversity of constrained three-dimensional peptide scaffolds present in these venoms that might be useful for bioengineering of drug leads. Expert opinion: Like most arthropod predators, centipede venoms are rich in peptides that target neuronal ion channels and receptors, but it is also becoming increasingly apparent that many of these peptides have novel or unexpected pharmacological properties with potential applications in drug discovery and development.

Extracting venom from small species is usually challenging. We describe here an affordable and versatile electrical venom extractor based on the Arduino(®) Mega 2560 Board, which is designed to extract venom from arthropods and other small animals. The device includes fine tuning of stimulation time and voltage. It was used to collect venom without apparent deleterious effects, and characterized for the first time the venom of Zoropsis spinimana, a common spider in French Mediterranean regions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Members of arachnida, such as spiders and scorpions, commonly produce venom with specialized venom glands, paralyzing their prey with neurotoxins that specifically target ion channels. Two well-studied motifs, the disulfide-directed hairpin (DDH) and the inhibitor cystine knot motif (ICK), are both found in scorpion and spider toxins. As arachnids, ticks inject a neurotoxin-containing cocktail from their salivary glands into the host to acquire a blood meal, but peptide toxins acting on ion channels have not been observed in ticks. Here, a new neurotoxin (ISTX-I) that acts on sodium channels was identified from the hard tick Ixodes scapularis and characterized. ISTX-I exhibits a potent inhibitory function with an IC50 of 1.6 μM for sodium channel Nav1.7 but not other sodium channel subtypes. ISTX-I adopts a novel structural fold and is distinct from the canonical ICK motif. Analysis of the ISTX-I, DDH and ICK motifs reveals that the new ISTX-I motif might be an intermediate scaffold between DDH and ICK, and ISTX-I is a clue to the evolutionary link between the DDH and ICK motifs. These results provide a glimpse into the convergent evolution of neurotoxins from predatory and blood-sucking arthropods.

In eusocial insects like Bald-faced hornets, Dolichovespula maculata, nest defense is essential because nests contain a large number of protein-rich larvae and pupae, and thus are attractive to nest predators. Our objectives were to investigate whether D. maculata exhibit pheromone-mediated nest defense, and to identify and field test any pheromone components. We tested for pheromone-mediated nest defense behavior of D. maculata by placing a paired box-apparatus near the entrance of D. maculata nests, and treating both boxes with a solvent control, or one of the two boxes with a solvent control and the other with either venom sac extract, the putative source of nest defense pheromone, or synthetic pheromone. The sound impulses caused by nest mates attempting to sting or strike the boxes were recorded for 3 min. Compared to the double-control treatment, the number of strikes increased 27-fold when one of the two boxes was treated with venom sac extract, providing evidence for an alarm response. The box treated with venom sac extract also induced a significantly greater proportion of strikes than the corresponding control box, providing evidence for a target-oriented response. Analyzing venom sac extract by gas chromatographic-electroantennographic detection (GC-EAD) and GC-mass spectrometry resulted in the identification of seven candidate pheromone components: (a) dimethylaminoethanol, (b) dimethylamino ethyl acetate, (c) 2,5-dimethylpyrazine, (d) N-3-methylbutylacetamide, (e) 2-heptadecanone, (f) (Z)-8-heptadecen-2-one, and (g) (Z)-10-nonadecen-2-one. Testing in paired-box bioassays blends of the nitrogen-containing volatile components a-d, the less volatile ketones e-g, or both (a-g), indicated that a-d primarily have an alarm function. The ketones e-g, in contrast, induced target-oriented responses, possibly marking the box, or potential nest predators, for guided and concerted attacks, or enhancing the alarm-inducing effect of the volatile pheromone components, as shown in honey bees. Comparing the behavioral effects of venom sac extract, blends a-d, e-g, and a-g, venom sac extract was most effective in triggering the full complement of alarm and target-oriented responses. These comparisons further suggested that a component is missing in the group of components that triggers the alarm rather than the target-oriented response.

Venomous animals have evolved with sophisticated bio-chemical strategies to arrest prey and defend themselves from natural predators. In recent years, peptide toxins from venomous animals have drawn considerable attention from researchers due to their surprising chemical, biochemical, and pharmacological diversity. Similar to other venomous animals, centipedes are one of the crucial venomous arthropods that have been used in traditional medicine for hundreds of years in China. Despite signifying pharmacological importance, very little is known about the active components of centipede venoms. More than 500 peptide sequences have been reported in centipede venomous glands by transcriptome analysis, but only a small number of peptide toxins from centipede has been functionally described. Like other venomous animals such as snakes, scorpions, and spiders, the venom of centipedes could be an excellent source of peptides for developing drugs for treatments as well as bio-insecticides for agrochemical applications. Although centipede venoms are yet to be adequately studied, the venom of centipedes as well as their components described to date, should be compiled to help further research. Therefore, based on previous reports, this review focusses on findings and possible therapeutic applications of centipede venoms as well as their components.

KCNE1 is a single-span transmembrane auxiliary protein that modulates the voltage-gated potassium channel KCNQ1. The KCNQ1/KCNE1 complex in cardiomyocytes exhibited slow activated potassium (I(ks)) currents. Recently, a novel 47-residue polypeptide toxin SSD609 was purified from Scolopendra subspinipes dehaani venom and showed I(ks) current inhibition. Here, chemically synthesized SSD609 was shown to exert I(ks) inhibition in extracted guinea pig cardiomyocytes and KCNQ1/KCNE1 current attenuation in CHO cells. The K(+) current attenuation of SSD609 showed decent selectivity among different auxiliary subunits. Solution nuclear magnetic resonance analysis of SSD609 revealed a distinctive three-helix conformation that was stabilized by a new disulfide bonding pattern as well as segregated surface charge distribution. Structure-activity studies demonstrated that negatively charged Glu19 in the amphipathic extracellular helix of KCNE1 was the key residue that interacted with SSD609. The distinctive three-helix centipede toxin SSD609 is known to be the first polypeptide toxin acting on channel auxiliary subunit KCNE1, which suggests a new type of pharmacological regulation for ion channels in cardiomyocytes.

The mosquito, Culex pipiens pallens (L.), is an important vector of encephalitis and filariasis in northern China. The control of these mosquitoes occurs primarily via the use of pyrethroid insecticides, such as deltamethrin. The widespread and improper application of pyrethroid has resulted in the evolution of pyrethroid resistance amongst many mosquito populations, including Cx. pipiens pallens. Previous studies using high-throughput transcriptome sequencing have identified that the venom allergen 5 gene is differentially expressed between deltamethrin-susceptible and deltamethrin-resistant Cx. pipiens pallens. In this study, quantitative real-time polymerase chain reaction analyses revealed that venom allergen 5 was significantly overexpressed in adult females of both deltamethrin-resistant laboratory populations and two field populations. The transcriptional level of venom allergen 5 in the laboratory populations was elevated as the levels of deltamethrin resistance increased. Full-length cDNAs of the venom allergen 5 gene were cloned from Cx. pipiens pallens, and contained an open reading frame of 765 bp, encoding a protein with 254 amino acids. The deduced amino acid sequence shared 100% identity with the ortholog in Culex quinquefasciatus Say. The overexpression of venom allergen 5 decreased the susceptibility of mosquito cells to deltamethrin, while knockdown of this gene by RNAi increased the susceptibility of mosquitoes to deltamethrin. This study provides the first evidence of the association between the venom allergen 5 gene and deltamethrin resistance in mosquitoes., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Stinging insect allergy is responsible for more than 10% of all cases of anaphylaxis. The potential culprit insects are diverse and vary with geography. The incidence of insect allergy is declining in some areas and increasing in others, possibly due to effects of climate change, introduction of species into new areas, outdoor recreational activities, and movement of human populations that brings insects into contact with a greater number of people. Flying Hymenoptera and imported fire ant stings are responsible for the majority of patients evaluated for insect anaphylaxis. The most efficient means of identifying allergy to insects is skin testing although falsely positive and negative results occur. The limitations of testing coupled with the natural temporal variability of allergic sensitivity complicate the interpretation of test results. The clinical history is of paramount importance to be certain that the test results are relevant; therefore, screening or testing before a history of a sting reaction is not advisable. Mast cell disorders are associated with severe anaphylaxis from insect stings and should be considered in affected subjects. Insect immunotherapy, using venoms for most insects and whole-body extracts for imported fire ants, is proven effective in reducing the likelihood of anaphylaxis due to subsequent stings from 40%-60% to less than 5%. Future clinical application of component testing or in vitro cellular tests, such as the basophil activation test, may improve optimal choices for immunotherapy., (Published by Elsevier Inc.)